The field of this invention is cell-based drug screens for regulators of the expression of the obese gene.
Satiety in vertebrates is controlled by a blood-borne hormone encoded by the obese (Ob) or leptin gene. Homozygous recessive mutations of the Ob gene (ob/ob) lead to the gross expansion of adipose tissue. Since animals lacking a functional Ob gene become phenotypically obese, it has been predicted that the Ob gene product plays a central role in energy homeostasis and appetite suppression.
The Ob gene has recently been cloned, facilitating molecular characterization of its encoded protein. The Ob gene product, termed leptin, is a secreted polypeptide produced by adipose tissue. Fat tissue accumulates in response to the intake of excess energy stores, becoming grossly expanded in animals lacking either functional leptin or its putative receptor. Under such circumstances, expression of the Ob gene is markedly elevated. These observations give evidence of a feedback loop responsible for controlling vertebrate energy balance. Adipose tissue subsides under conditions of food deprivation, resulting in a reduced level of leptin production and a corresponding increase in appetite. In the well-fed state, excess energy stores accumulate in adipose tissue. Upon maturation and expansion, adipocytes activate expression of the Ob gene, whose product then serves to quell satiety and stimulate metabolic activity.
Several lines of evidence have indicated that leptin production may be regulated at the level of transcription of its encoding gene. Adipose tissue derived from homozygous Ob-defective animals contains appreciably higher levels of leptin mRNA than that of either heterozygous or wild-type controls. Increased levels of Ob mRNA have also been observed in obese humans. Furthermore, expression of the Ob gene is elevated in response to insulin and other blood borne hormones involved in energy homeostasis. Finally, thiazolinedione derivatives, used to reduced insulin resistance in non-insulin dependent diabetes, have been shown to reduce Ob gene expression. These observations provide evidence that expression of the Ob gene is sensitively balanced with respect to the supply of metabolic energy stores as well as the hormonal factors responsible for controlling energy homeostasis.
RELEVANT LITERATURE
Zhang, Y., et al. (1994) Nature 372, 425-432 report the cloning of the Ob gene. de la Brousse et al. (1996) PNAS 93, 4096-4101, He et al. (1995) J. Biol Chem 270, 28887-28891, Hwang et al. (1996) PNAS 93, 873-877, Gong et al. (1996) J. Biol Chem 271, 3971-3974, report the identification of transcriptional control elements of the ob gene.
The invention provides methods and compositions for screening for agents which regulate the level of ob gene expression. Such agents find use in modulating a wide variety of physiological manifestations of ob gene expression including satiety, fat metabolism, disease states such as obesity, type II diabetes mellitus, etc.
The subject assays are cell-based and generally involve (a) contacting a mammalian adipocyte comprising a mutant of a native ob allele encoding a reporter of ob gene expression, wherein the expression of the reporter is under the control of the native gene expression regulatory sequences of the native ob allele, with a candidate agent under conditions whereby but for the presence of the agent, the reporter is expressed at a first expression level; and, (b) measuring the expression of the reporter to obtain a second expression level, wherein a difference between the first and second expression levels indicates that the candidate agent modulates ob gene expression.
The mutant may result from replacement of a portion of said native ob allele with a sequence encoding said reporter. For example, the adipocyte may be a progeny of a genetic knock-in cell made by homologous recombination of the native ob allele with a transgene comprising a sequence encoding the reporter flanked by flanking sequences capable of effecting the homologous recombination of the transgene with the native ob allele, a positive selectable marker positioned inside the flanking sequences and a negative selectable marker positioned outside the flanking sequences. The adipocyte may be a primary adipocyte residing in or isolated from an animal transgenic in the mutant or derive from a cultured cell line transgenic in the mutant. Preferred methods are ex-vivo cell-based transcription assays using adipocytes from transgenic animals having a xe2x80x9cknock-inxe2x80x9d luciferase reporter at least one ob allele.
The invention also encompasses mammalian adipocytes and mammals transgenic in a mutant of a native ob allele encoding a reporter of ob gene expression, wherein the expression of the reporter is under the control of the gene expression regulatory sequences of the native ob allele, genetic knock in vectors for making such animals and cells and methods of making and using such vectors, cells and animals.